The central nervous system (CNS) is made up of a large number of interacting neuronal systems and these, in turn, are comprised of heterogeneous cell populations. Analysis of cellular diversity within a given system has been based largely on the presence of morphological and protein markers. However, individual cells may differ functionally based on their localization, synaptic and glial interactions and the cadre of genes which are expressed. These factors combine to determine the cellular and functional identity of individual cells within the CNS. This interaction is so complex that the variety of cellular identities is certainly far greater than has been recognized to date using established methods.
The ability to experimentally manipulate single cells using whole cell and patch clamp techniques has provided a specificity and sensitivity of analysis which has permitted the characterization of the electrophysiological properties of single cells. While tremendous strides have been made on the electrophysiological front, little progress has been made in understanding the regulation of mRNA levels in individual cells. The limitation has been that most biochemical and molecular biological studies have used complex neural systems as sources of RNA. These include brain slices or dissected tissue samples and heterogeneous populations of cells grown in culture. Such preparations suffer from the large amount of tissue that is required for analysis, thereby making analysis of cellular specificity problematic. While in situ hybridization can be used to assess gene expression in individual fixed cells, this approach is limited in its ability to depict accurately the entire mRNA population of the cell. Furthermore, it has not been possible to obtain electrophysiological recordings from the individual cell of interest prior to the in situ hybridization. Examining the entire mRNA population of a cell is an important aspect of cellular responsiveness since mRNA level changes often dictate changes in protein concentrations. If mRNA levels within the cell can be detected, then it may be possible to predict protein production and cellular responsiveness to various exogenous stimuli.
The amount of DNA within a single cell is estimated to be between 0.1 and 1 picograms and, as such, is difficult to manipulate experimentally. The most commonly employed amplification procedure is the polymerase chain reaction (PCR). To utilize this technology, one must first make cDNA from RNA isolated from a single cell using reverse transcriptase followed by the addition of a primer site to the 3'-end of the first strand cDNA, most commonly by the action of terminal transferase. The amplification process utilizes this added sequence and the poly-A tail of second strand cDNA as priming sites for the amplification process. PCR has been utilized to amplify cDNA populations encoding mRNA from as few as 1000 cells. Tam et al., Nuc. Acids Res., 17:1269 (1989); Belyavski et al., Nuc. Acids Res., 17:2919-2932 (1989).
PCR technology, however, suffers from several drawbacks which limit its utility for studies of single cells. PCR works best when small regions of a few hundred nucleotides are being amplified. When larger cDNAs are amplified, there is a disproportionate decrease in the level of amplification such that longer cDNAs are not amplified at the same rate as shorter cDNAs. In addition, the error rate of the enzyme most commonly used for PCR, namely Taq polymerase, is high enough such that errors are estimated to be incorporated once per 1,000 bases of incorporation. With such an error rate, it is certain that most PCR-amplified cDNAs will contain several erroneous bases. While these problems can be dealt with if anticipated, it is not possible to easily address the difference in the efficiency of the amplification of different cDNA molecules. Even a small difference in efficiency will result in several thousand fold differential representation of these cDNAs in the cDNA population after as few as 30 rounds of amplification. It is possible that with technological improvements in PCR amplifications, it could become a useful approach in the routine isolation of individual cDNAs from single cells of the nervous system. However, these technological problems currently limit the overall usefulness of PCR in the study of gene expression in single cells.
The technique of amplified, antisense RNA (aRNA) synthesis (VanGelder et al., Proc. Natl. Acad. Sci. USA, 87:1663-1667 (1990)) circumvents many of these problems by facilitating the linear amplification of large mRNAs with few errors. The first step is the synthesis of an oligo-d(T) primer that is extended at the 5'-end with a T7 RNA polymerase promoter. This oligonucleotide can be used to prime the poly-A+-mRNA population for cDNA synthesis. After the first strand cDNA is synthesized, the second-strand cDNA is made using either "Gubler-Hoffman" for RNA in solution (Gubler, V. and Hoffman, B. Genet., 25:263-269 (1983), or "hairpinning" for tissue sections (Maniatis et al., Cell, 15:687-701 (1978)). This is followed by a brief S1-nuclease treatment and "blunt-ending" with T4-DNA polymerase. The cDNA is now ready for amplification using the T7 RNA polymerase promoter (Melton et al., Nuc. Acids Res., 12:7035-7056 (1984); Nielsen, D. A. and Shapiro, D. J., Nuc. Acids Res., 14:5936-5940 (1986)) to direct the synthesis of RNA. The RNA made using this technique is antisense to the poly-A+ RNA and can be used directly as a probe, or it can be cloned into plasmid or phage vectors using standard techniques (Sambrook et al., Molecular Cloning, 2nd Ed., Cold Spring Harbor Press, 1989).
U.S. Pat. No. 5,021,335 (Tecott et al.) discloses in situ transcription for cDNA synthesis in cells and tissues. Fixed tissue sections are contacted with a nucleotide primer under hybridizing conditions and then contacted with reverse transcriptase under primer-extension conditions. Labeling with radioactive nucleotides to follow primer extension in the tissue section may be performed using standard techniques. The transcript may then be isolated by elution and used in cloning, expression, etc.
Although aRNA provides a means for amplifying RNA populations, it is extremely difficult to isolate RNA from a single cell. The primary obstacle to the isolation of RNA from a single cell is the tendency of RNA to nonspecifically interact with plastic and glass.